Chemical arrays and methods of producing the same

ABSTRACT

Methods and devices for fabricating a chemical array are provided. Embodiments include determining a chemical array layout in which each feature in the layout has a size that is chosen based on its composition and fabricating a chemical array according to the chemical array layout. In certain embodiments, at least two features of an array fabricated according to the subject methods are of different sizes. Embodiments also include chemical arrays having features of different sizes, e.g., fabricated according to the subject methods. Also provided are embodiments that include fluid deposition devices capable of fabricating chemical arrays having features of different sizes, e.g., for use in practicing the subject methods. Algorithms present on computer readable mediums for use in practicing the subject methods may also be provided in certain embodiments. Embodiments of the subject invention may also include systems and kits for use in practicing the subject methods.

FIELD OF THE INVENTION

The present invention relates to chemical arrays.

BACKGROUND OF THE INVENTION

Chemical arrays such as biopolymer arrays (for example polynucleotidearray such as DNA or RNA arrays), are arrays of binding agents (ligands)and are known and are used, for example, as diagnostic or screeningtools, including, but not limited to, gene expression analysis, drugscreening, nucleic acid sequencing, mutation analysis, and the like.These binding agent or ligand arrays include a plurality of bindingagents positioned on a solid support surface in the form of an array orpattern.

Where the ligands of the arrays are polymeric, e.g., as is the case withnucleic acid and polypeptide arrays, there are two main ways ofproducing such arrays, i.e., via in-situ synthesis in which thepolymeric ligand is grown on the surface of the substrate in a step-wisefashion and via deposition of the full ligand, e.g., a pre-synthesizednucleic acid/polypeptide, cDNA fragment, etc., onto the surface of thearray.

In many instances, it may be desirable for an array to have features ofdifferent sizes. Of particular interest would be the ability tofabricate such arrays in a high-throughput manner.

Accordingly, there continues to be an interest in the development ofmethods and devices capable of fabricating a biopolymeric array withfeatures of different sizes, i.e., that enable control of the size ofeach feature of an array, e.g., in a high throughput manner. Ofparticular interest are such methods and devices that enable control ofeach feature on a per probe basis.

SUMMARY OF THE INVENTION

Methods and devices for fabricating a chemical array are provided.Embodiments of the subject methods include determining a chemical arraylayout in which each feature in the layout has a size that is chosenbased on its composition and fabricating a chemical array according tothe chemical array layout. In certain embodiments, at least two featuresof an array fabricated according to the subject methods are of differentsizes. Embodiments also include chemical arrays having features ofdifferent sizes, e.g., fabricated according to the subject methods. Alsoprovided are embodiments that include fluid deposition devices capableof fabricating chemical arrays having features of different sizes, e.g.,for use in practicing the subject methods. Algorithms present oncomputer readable mediums for use in practicing the subject methods mayalso be provided in certain embodiments. Embodiments of the subjectinvention may also include systems and kits for use in practicing thesubject methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary embodiment of a waveform that may be employedin the practice if the subject invention.

FIG. 2 shows a block diagram of an exemplary embodiment of the subjectinvention.

FIG. 3 shows an exemplary embodiment of an apparatus which may beemployed in the practice of the subject invention.

FIG. 4 shows an exemplary embodiment of an array reader which may beemployed to read arrays produces according to the subject methods.

FIG. 5 shows an exemplary embodiment of an array assembly that may beproduced according to the subject methods.

FIG. 6 shows an enlarged view of a portion of FIG. 8 showing spots orfeatures of differing sizes.

FIGS. 7-8 show exemplary embodiments of array patterns having featuresof different sizes in accordance with the subject invention.

DEFINITIONS

A “biopolymer” is a polymer of one or more types of repeating units.Biopolymers are typically found in biological systems and include, butare not limited to, polysaccharides (such as carbohydrates), andpeptides (which term is used to include polypeptides, and proteinswhether or not attached to a polysaccharide) and polynucleotides as wellas their analogs, such as those compounds composed of or containingamino acid analogs or non-amino acid groups, or nucleotide analogs ornon-nucleotide groups. This includes polynucleotides in which theconventional backbone has been replaced with a non-naturally occurringor synthetic backbone, and nucleic acids (or synthetic or naturallyoccurring analogs) in which one or more of the conventional bases hasbeen replaced with a group (natural or synthetic) capable ofparticipating in Watson-Crick type hydrogen bonding interactions orWobble interactions. Polynucleotides include single or multiple strandedconfigurations, where one or more of the strands may or may not becompletely aligned with another. A “nucleotide” refers to a sub-unit ofa nucleic acid and has a phosphate group, a 5 carbon sugar and anitrogen containing base, as well as functional analogs (whethersynthetic or naturally occurring) of such sub-units which in the polymerform (as a polynucleotide) can hybridize with naturally occurringpolynucleotides in a sequence specific manner analogous to that of twonaturally occurring polynucleotides. For example, a “biopolymer”includes DNA (including cDNA), RNA, oligonucleotides, and PNA and otherpolynucleotides as described in U.S. Pat. No. 5,948,902 and referencescited therein (all of which are incorporated herein by reference),regardless of the source. An “oligonucleotide” generally refers to anucleotide multimer of about 10 to 100 nucleotides in length, while a“polynucleotide” includes a nucleotide multimer having any number ofnucleotides.

A “biomonomer” references a single unit, which can be linked with thesame or other biomonomers to form a biopolymer (for example, a singleamino acid or nucleotide with two linking groups one or both of whichmay have removable protecting groups). A biomonomer fluid or biopolymerfluid reference a liquid containing either a biomonomer or biopolymer,respectively (typically in solution).

A chemical “array”, unless a contrary intention appears, includes anyone, two or three-dimensional arrangement of addressable regions bearinga particular chemical moiety or moieties (for example, biopolymers suchas polynucleotide sequences) associated with that region. Each regionmay extend into a third dimension in the case where the substrate isporous while not having any substantial third dimension measurement(thickness) in the case where the substrate is non-porous. An array is“addressable” in that it has multiple regions of different moieties (forexample, different polynucleotide sequences) such that a region (a“feature” or “spot” of the array) at a particular predetermined location(an “address”) on the array will detect a particular target or class oftargets (although a feature may incidentally detect non-targets of thatfeature). Any given substrate may carry one, two, four or more arraysdisposed on a front surface of the substrate. Depending upon the use,any or all of the arrays may be the same or different from one anotherand each may contain multiple spots or features.

An array may contain one or more, including more than two, more thanten, more than one hundred, more than one thousand, more ten thousandfeatures, or even more than one hundred thousand features, in an area ofless than 20 cm² or even less than 10 cm², e.g., less than about 5 cm²,including less than about 1 cm², less than about 1 mm², e.g., 100 β²; oreven smaller. In certain embodiments, an array may cover an area asgreat as about 230 cm² or more, e.g., as great as about 930 cm² or more.By “feature” or “spot”, used interchangeably, is meant a polymer, i.e.,binding agent, present as a composition of multiple copies of thepolymer on an array substrate surface. The multiple copies may be in anyshape, including round and non-round shapes.

For example, features may have widths (that is, diameter, for a roundspot) in the range from about 10 μm to about 1.0 cm. In otherembodiments each feature may have a width in the range of about 1.0 μmto about 1.0 mm, usually about 5.0 μm to about 500 μm, and more usuallyabout 10 μm to about 200 μm. Non-round features may have area rangesequivalent to that of circular features with the foregoing width(diameter) ranges. At least some, or all, of the features are ofdifferent compositions (for example, when any repeats of each featurecomposition are excluded the remaining features may account for at least5%, 10%, 20%, 50%, 95%, 99% or 100% of the total number of features).Inter-feature areas will typically (but not essentially) be presentwhich do not carry any nucleic acids (or other biopolymer or chemicalmoiety of a type of which the features are composed). It will beappreciated though, that the inter-feature areas, when present, could beof various sizes and configurations.

Each array may cover an area of less than 200 cm², or even less than 50cm², 5 cm², 1 cm², 0.5 cm², or 0.1 cm². In certain embodiments, thesubstrate carrying the one or more arrays will be shaped generally as arectangular solid (although other shapes are possible), having a lengthof more than 4 mm and less than 150 mm, usually more than 4 mm and lessthan 80 mm, more usually less than 20 mm; a width of more than 4 mm andless than 150 mm, usually less than 80 mm and more usually less than 20mm; and a thickness of more than 0.01 mm and less than 5.0 mm, usuallymore than 0.1 mm and less than 2 mm and more usually more than 0.2 andless than 1.5 mm, such as more than about 0.8 mm and less than about 1.2mm. With arrays that are read by detecting fluorescence, the substratemay be of a material that emits low fluorescence upon illumination withthe excitation light. Additionally in this situation, the substrate maybe relatively transparent to reduce the absorption of the incidentilluminating laser light and subsequent heating if the focused laserbeam travels too slowly over a region. For example, the substrate maytransmit at least 20%, or 50% (or even at least 70%, 90%, or 95%), ofthe illuminating light incident on the front as may be measured acrossthe entire integrated spectrum of such illuminating light oralternatively at 532 nm or 633 nm. In certain embodiments, the substratemay include a mirrored surface.

In the case of an array, the “target” will be referenced as a moiety ina mobile phase (typically fluid), to be detected by probes (“targetprobes”) which are bound to the substrate at the various regions.However, either of the “target” or “target probes” may be the one whichis to be evaluated by the other (thus, either one could be an unknownmixture of polynucleotides to be evaluated by binding with the other).

An “array layout” or “array characteristics”, refer to one or morephysical, chemical or biological characteristics of the array, such asfeature positioning, one or more feature dimensions such as featuresize, density, and the like, some indication of an identity or function(for example, chemical or biological) of a moiety at a given location,how the array should be handled (for example, conditions under which thearray is exposed to a sample, or array reading specifications orcontrols following sample exposure), and the like.

The term “hybridization” as used herein refers to binding betweencomplementary or partially complementary molecules, for example asbetween the sense and anti-sense strands of double-stranded DNA. Suchbinding is commonly non-covalent binding, and is specific enough thatsuch binding may be used to differentiate between highly complementarymolecules and others less complementary. Examples of highlycomplementary molecules include complementary oligonucleotides, DNA,RNA, and the like, which comprise a region of nucleotides arranged inthe nucleotide sequence that is exactly complementary to a probe;examples of less complementary oligonucleotides include ones withnucleotide sequences comprising one or more nucleotides not in thesequence exactly complementary to a probe oligonucleotide. “Hybridizing”and “binding”, with respect to polynucleotides, are usedinterchangeably.

An “array assembly” may be one or more arrays plus only a substrate onwhich the one or more arrays are deposited, although the assembly may bein the form of a package which includes other elements (such as ahousing with a chamber). Specifically, an array assembly at leastincludes a substrate having at least one array thereon.

When one item is indicated as being “remote” from another, this isreferenced that the two items are at least in different buildings, andmay be at least one mile, ten miles, or at least one hundred milesapart. “Communicating” information references transmitting the datarepresenting that information as electrical signals over a suitablecommunication channel (for example, a private or public network).“Forwarding” an item refers to any means of getting that item from onelocation to the next, whether by physically transporting that item orotherwise (where that is possible) and includes, at least in the case ofdata, physically transporting a medium carrying the data orcommunicating the data.

A “chamber” references an enclosed volume (although a chamber may beaccessible through one or more ports).

It will also be appreciated that throughout the present application,that words such as “front”, “back”, “top”, “upper”, and “lower” are usedin a relative sense only.

“Fluid” is used herein to reference a liquid.

“May” refers to optionally. Any recited method can be carried out in theordered sequence of events as recited, or any other logically possiblesequence. “Optional” or “optionally” means that the subsequentlydescribed circumstance may or may not occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

A “fluid drop deposition device” and analogous terms refers broadly toany device which can dispense drops in the formation of an array andincludes, but is not limited to, pulse jet devices. “Pulse jets” operateby delivering a pulse of pressure (such as by a piezoelectric orthermoelectric element) to liquid adjacent an outlet or orifice suchthat a drop will be dispensed therefrom.

“Continuous” in reference to an area on the substrate surface referencesan area which is uninterrupted by any gaps within that area. Thedistinct features of an array may then be formed on such a continuousarea.

“Probe density” is a shorthand way of referring to the number of linkermolecules or probe molecules per unit area within a feature. This termthen is used interchangeably with, and has the same meaning as “featureprobe density”. Thus, any interfeature areas which are essentiallydevoid of the probe are not taken into consideration in determining aprobe density. “Probe density” in a region then, is distinct andindependent of feature density (which is the number of features per unitarea).

Different feature sizes with respect to the sizes of at least twofeatures refers to sizes of the referenced features that differ by morethan about 1% or more, e.g., more than about 5%, e.g., more than 10%,15%, 20% or 50% or more. By “feature size” is meant a characteristiclength scale of a feature. For example, a characteristic length scalemay be width (diameter for a round feature) or the like.

The terms “target” “target molecule” “target biomolecule” and “analyte”are used herein interchangeably and refer to a known or unknown moleculein or suspected of being in a sample. A target is one that will bind,e.g., hybridize, to a probe on a substrate surface if the targetmolecule and the molecular probe are complementary, e.g., if theycontain complementary regions, i.e., if they are members of a specificbinding pair.

The term “probe” as used herein refers to a molecule of known identityadherent to a substrate.

“Probe copies” refers to exact copies of a given probe.

The term “hybridization solution” or “hybridization reagent” used hereininterchangeably refers to a solution suitable for use in a hybridizationreaction.

A “linking layer” bound to the surface may, for example, be less than200 angstroms or even less than 10 angstroms in thickness (or less than8, 6, or 4 angstroms thick). Such layer may have a polynucleotide,protein, nucleoside or amino acid minimum binding affinity of 104 to 106units/μ². Layer thickness may be evaluated using UV or X-rayelipsometry.

The term “stringent assay conditions” as used herein refers toconditions that are compatible to produce binding pairs of nucleicacids, e.g., surface bound and solution phase nucleic acids, ofsufficient complementarity to provide for the desired level ofspecificity in the assay while being less compatible to the formation ofbinding pairs between binding members of insufficient complementarity toprovide for the desired specificity. Stringent assay conditions are thesummation or combination (totality) of both hybridization and washconditions.

A “stringent hybridization” and “stringent hybridization washconditions” in the context of nucleic acid hybridization (e.g., as inarray, Southern or Northern hybridizations) are sequence dependent, andare different under different experimental parameters. Stringenthybridization conditions that can be used to identify nucleic acidswithin the scope of the invention can include, e.g., hybridization in abuffer comprising 50% formamide, 5×SSC, and 1% SDS at 42° C., orhybridization in a buffer comprising 5×SSC and 1% SDS at 65° C., bothwith a wash of 0.2×SSC and 0.1% SDS at 65° C. Exemplary stringenthybridization conditions can also include a hybridization in a buffer of40% formamide, 1 M NaCl, and 1% SDS at 37° C., and a wash in 1×SSC at45° C. Alternatively, hybridization to filter-bound DNA in 0.5 M NaHPO4,7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. can be employed. Yet additional stringenthybridization conditions include hybridization at 60° C. or higher and3×SSC (450 mM sodium chloride/45 mM sodium citrate) or incubation at 42°C. in a solution containing 30% formamide, 1M NaCl, 0.5% sodiumsarcosine, 50 mM MES, pH 6.5. Those of ordinary skill will readilyrecognize that alternative but comparable hybridization and washconditions can be utilized to provide conditions of similar stringency.

In certain embodiments, the stringency of the wash conditions that setforth the conditions which determine whether a nucleic acid isspecifically hybridized to a surface bound nucleic acid. Wash conditionsused to identify nucleic acids may include, e.g.: a salt concentrationof about 0.02 molar at pH 7 and a temperature of at least about 50° C.or about 55° C. to about 60° C.; or, a salt concentration of about 0.15M NaCl at 72° C. for about 15 minutes; or, a salt concentration of about0.2×SSC at a temperature of at least about 50° C. or about 55° C. toabout 60° C. for about 15 to about 20 minutes; or, the hybridizationcomplex is washed twice with a solution with a salt concentration ofabout 2×SSC containing 0.1% SDS at room temperature for 15 minutes andthen washed twice by 0.1×SSC containing 0.1% SDS at 68° C. for 15minutes; or, equivalent conditions. Stringent conditions for washing canalso be, e.g., 0.2×SSC/0.1% SDS at 42° C.

A specific example of stringent assay conditions is rotatinghybridization at 65° C. in a salt based hybridization buffer with atotal monovalent cation concentration of 1.5 M (e.g., as described inU.S. patent application Ser. No. 09/655,482 filed on Sep. 5, 2000, thedisclosure of which is herein incorporated by reference) followed bywashes of 0.5×SSC and 0.1×SSC at room temperature.

Stringent assay conditions are hybridization conditions that are atleast as stringent as the above representative conditions, where a givenset of conditions are considered to be at least as stringent ifsubstantially no additional binding complexes that lack sufficientcomplementarity to provide for the desired specificity are produced inthe given set of conditions as compared to the above specificconditions, where by “substantially no more” is meant less than about5-fold more, typically less than about 3-fold more. Other stringenthybridization conditions are known in the art and may also be employed,as appropriate.

The term “ligand” as used herein refers to a moiety that is capable ofcovalently or otherwise chemically binding a compound of interest.Ligands may be naturally-occurring or manmade. Examples of ligandsinclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones,opiates, steroids, peptides, enzyme substrates, cofactors, drugs,lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, andproteins.

The term “receptor” as used herein is a moiety that has an affinity fora ligand. Receptors may be naturally-occurring or manmade. They may beemployed in their unaltered state or as aggregates with other species.Receptors may be attached, covalently or noncovalently, to a bindingmember, either directly or via a specific binding substance. Examples ofreceptors include, but are not restricted to, antibodies, cell membranereceptors, monoclonal antibodies and antisera reactive with specificantigenic determinants, viruses, cells, drugs, polynucleotides, nucleicacids, peptides, cofactors, lectins, sugars, polysaccharides, cellularmembranes, and organelles. Receptors are sometimes referred to in theart as anti-ligands. As the term receptors is used herein, no differencein meaning is intended. A “Ligand Receptor Pair” is formed when twomolecules have combined through molecular recognition to form a complex.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid form,containing or suspected of containing one or more components (targets)of interest.

A “computer-based system” refers to the hardware means, software means,and data storage means used to analyze the information of the presentinvention. The minimum hardware of computer-based systems as they relateto the present invention include a central processing unit (CPU), inputmeans, output means, and data storage means. A skilled artisan canreadily appreciate that any one of the currently availablecomputer-based system are suitable for use in the present invention. Thedata storage means may include any manufacture comprising a recording ofthe present information as described above, or a memory access meansthat can access such a manufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats may be used forstorage, e.g. word processing text file, database format, etc.

A “processor” references any hardware and/or software combination thatwill perform the functions required of it. For example, any processorherein may be a programmable digital microprocessor such as available inthe form of an electronic controller, mainframe, server or personalcomputer (desktop or portable). Where the processor is programmable,suitable programming can be communicated from a remote location to theprocessor, or previously saved in a computer program product (such as aportable or fixed computer readable storage medium, whether magnetic,optical or solid state device based). For example, a magnetic medium oroptical disk may carry the programming, and can be read by a suitablereader communicating with each processor at its corresponding station.

“Activation signal” refers to the electrical or other analogous energyprovided to an ejector to activate the ejector.

“Waveform” refers to the shape of an activation signal which may beillustrated graphically by plotting the values of voltage against time.

DETAILED DESCRIPTION OF THE INVENTION

Methods and devices for fabricating a chemical array are provided.Embodiments of the subject methods include determining a chemical arraylayout in which each feature in the layout has a size that is chosenbased on its composition and fabricating a chemical array according tothe chemical array layout. In certain embodiments, at least two featuresof an array fabricated according to the subject methods are of differentsizes. Embodiments also include chemical arrays having features ofdifferent sizes, e.g., fabricated according to the subject methods. Alsoprovided are embodiments that include fluid deposition devices capableof fabricating chemical arrays having features of different sizes, e.g.,for use in practicing the subject methods. Algorithms present oncomputer readable mediums for use in practicing the subject methods mayalso be provided in certain embodiments. Embodiments of the subjectinvention may also include systems and kits for use in practicing thesubject methods.

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention.

The figures shown herein are not necessarily drawn to scale, with somecomponents and features being exaggerated for clarity.

Methods of Producing a Chemical Array

As noted above, embodiments of the subject invention include methods forfabricating a chemical array such as a biopolymeric array. Specifically,the subject invention provides methods for fabricating a chemical arraythat enable-precise control over the sizes of each feature of thechemical array. Accordingly, chemical arrays may be fabricated accordingto the subject invention having one or more features, including all ofthe features, of different sizes. In certain embodiments, features ofdiffering sizes may be adjacent each other, e.g., in the same column orrow of the array, such that there are not intervening featurestherebetween. In certain embodiments, fluid for fabricating differentsized features of an array may be dispensed from the same orifice of adrop deposition apparatus, e.g., adjacent features. For example, theamount of fluid dispensed from an orifice of a drop deposition devicemay be changed, e.g., by modulating the activation signal provided tothe orifice ejector associated with the orifice, during the fabricationprocess without disruption of the fabrication process, to dispensedifferent volumes of fluid from the orifice, thereby fabricatingfeatures of different sizes from the same orifice.

As noted above and which will be described in greater detail below, thechemical arrays of the subject invention are, in the broadest sense,arrays of polymeric or biopolymeric ligands or molecules, i.e., bindingagents or probes, where the polymeric binding agents may be any of:peptides, proteins, nucleic acids, polysaccharides, synthetic mimeticsof such biopolymeric binding agents, etc. In many embodiments ofinterest, the arrays are arrays of nucleic acids, includingoligonucleotides, polynucleotides, cDNAs, mRNAs, synthetic mimeticsthereof, and the like. As described in greater detail below, thechemical arrays of the subject invention may be employed in arrayassays, e.g., hybridization assays, in which the arrays are contactedwith a sample containing, or suspected of containing, one or moretargets of interest. Once contacted, and further processed if required,any probe/target binding complexes present on the array may be detectedto provide information about the presence of the one or more targets inthe sample.

In general, the subject methods employ a fluid deposition device thatincludes at least one deposition head (printhead) having at least onereservoir associated with at least one orifice (also referred to as anozzle), through which fluid is ejected. An orifice of the depositionhead includes an ejector, which, when activated, causes fluid (e.g.,probe precursor (e.g., phosphoramidite), activator fluid (e.g.,tetrazole activator) and the like) to be expelled from the orifice ontoan array substrate.

Embodiments of the subject methods include controlling the amount offluid (i.e., the droplet size) dispensed from the one or more orificesof the deposition head, e.g., by modulating the applied activationsignal provided to each respective ejector, e.g., prior to each ejectionof fluid from the deposition head, wherein the activation signalprovided to an ejector is directly related to the amount of fluidejected from a respective orifice and thus is directly related to thesize of each feature. Embodiments the subject invention includemodulating the applied waveform provided to each ejector to control theamount of fluid dispensed from the one or more orifices.

It is to be understood that the term “modulation” is used hereinbroadly. For example, modulation of, e.g., an applied activation signal,may be with respect to amplitude, pulse length, frequency, and the like.

Embodiments of the subject methods include modulating an applied voltageto each ejector, thus enabling the adjustment of the volume of fluiddelivered from a deposition head for each array feature, i.e., providedynamic fluid volume adjustments. In many embodiments, a deposition heademployed in the practice of the subject methods includes a plurality ofreservoirs and corresponding orifices, each orifice having a respectiveejector. Accordingly, embodiments of the subject invention includecontrolling the amount of fluid dispensed from each orifice of thedeposition head, e.g., by modulating the applied activation signal toeach ejector, e.g., prior to each ejection of fluid from a correspondingorifice.

Embodiments of the subject invention enable a chemical array to beprepared or “customized” at least with respect to each feature size ofthe prepared array. This customization may be accomplished bydetermining a chemical array layout in which each feature in thechemical array layout has a size that is chosen based on itscomposition, and fabricating a chemical array according to thebiopolymeric array layout. As described in greater detail below, onemanner in which this may be accomplished is by providing variousactivation signals to different ejectors of a fluid drop depositiondevice employed to fabricate the array according to the array layout. Inother words, precisely controlling the particular activation signalprovided to each ejector of a deposition head enables customization ofthe size of each feature of an array. As the activation signal providedto an ejector is directly related to the amount of fluid ejected from anorifice associated with that particular orifice, ejectors capable ofbeing activated using differing signals to eject different amounts offluids therefrom provides precise control over the feature sizes of anarray is provided and thus enable fabrication of an array of features ofvarious sizes. Embodiments include providing signals with variouswaveforms to different ejectors of a fluid drop deposition deviceemployed to fabricate the array according to the array layout toaccomplish the above-described fabrication.

It may be desirable to vary the feature sizes of an array for a numberof reasons. For example, it may be advantageous to do so to customizeand/or optimize the array to a particular sample with which the array isdesigned to be used. For example, the abundance level of at least onetarget in a sample for which the array is designed to be used may beunknown and thus the subject invention may be employed to design, e.g.,optimize, the array features for that particular sample, e.g., in aniterative process. Analogously, an array having various feature sizesmay be employed to tailor the array with respect to the at leastsuspected (i.e., known or anticipated) abundance level of at least onetarget in a sample for which the array is designed to be used. In thismanner, the signal obtained from the binding of the target to therespective features may be customized or tailored to the amount oftarget present or suspected of being present in the sample. Accordingly,in certain embodiments using these customized arrays, features may befabricated that provide signal obtained from the resultant bindingcomplexes (feature/target binding complex) that is appropriate orcommensurate with limitations of the system, e.g., with respect tonoise, detection limit, etc.

As noted above, the ability to control the size of each feature of anarray is provided by the subject methods. That is, the subject methodsprovide the ability to customize the chemistry or feature size for eachfeature (e.g., for each synthesized base) on a per surface bound ligand,e.g., probe, basis (as opposed to a per print swath column or per entiresubstrate or entire substrate layer basis). As the subject methodsprovide features of different sizes on a per feature basis, faster arrayfabrication times for arrays with different features sizes may beobtained because separate printing swath columns are not required toproduce different feature sizes, as the feature size of each, individualfeature of an array may be controlled within each printing swath. By“swath column” is meant a complete column of features of an array on asubstrate surface or a complete pass of the fluid deposition device headacross the array substrate upon which fluid (e.g., a phosphoramidite,activator fluid, and the like) is deposited from the deposition head.For example, a swath may encompass a pass from a first side of thesubstrate to a second side.

Accordingly, a chemical array fabricated according to the subjectinvention may have multiple features of different sizes (or all of thefeatures sizes may be the same), but in any event the subject methodsenable control over the amount of fluid to be dispensed from eachorifice of a deposition head, e.g., prior to each ejector activationevent. For example, embodiments may include the fluid drop depositionfabrication of a multi-feature chemical array having a first feature ofa first size formed on a region of an array substrate and at least asecond feature of a second size formed on a region of the substrate,where the different feature sizes result at least in part from differentapplied activation signals to ejectors employed in the fabrication ofthe different sized features. The number of features of a chemical arrayof different sizes may vary depending on the particular array and mayrange from about 2 or more, e.g., may be at least about five, at leastabout ten, at least about one hundred, or at least about one or abouttwo thousand or more. Features of different sizes may be directlyadjacent one another in certain embodiments such that a first feature ofa first size may be directly next to (without any intervening features)a second feature of a second size.

As noted above, the subject methods employ a fluid drop depositiondevice to fabricate one or more arrays on an array substrate surface,where exemplary fluid drop deposition devices include, but are notlimited to, pulse jet deposition devices and the like. In general,embodiments of an apparatus employed in the subject invention mayinclude an optional substrate holder on which the substrate can bemounted, and a drop deposition system to deposit drops of the probes orprobe precursors. A processor may control the drop deposition system soas to contact fluid from the drop deposition system, e.g., differentprobes or probe precursors, with different locations on the substratesurface, and repeat this as needed, so as to form the array. Theprocessor may also receive an indication of the location of differentregions on a substrate to which fluid, e.g., probes or probe precursorswill bind, and controls the drop deposition system to form the arraywith features of different sizes. The processor may also control theamount of fluid dispensed from each orifice of the deposition head so asto control the size of each feature of the array, e.g., by providing awaveform to each ejector based on the feature size desired to befabricated.

Computer program products of the present invention may include acomputer readable storage medium having a computer program storedthereon which controls the apparatus to perform a method as describedherein. Any computer readable storage medium for any purpose herein mayinclude, for example, an optical or magnetic memory (such as a fixed orportable disk or other device), or a solid state memory.

In further describing the subject invention, an exemplary apparatus thatmay be employed in the practice of the subject methods is describedfirst to provide a foundation for the subject methods.

Fluid Drop Deposition Apparatuses

Any suitable fluid drop deposition device may be employed in thepractice of the subject invention. In general, such devices include adeposition head system that may contain one or more (for example two)heads mounted on the same head retainer (e.g., see head retainer 208 ofFIG. 3). Each such head may be of the type commonly used in an ink jettype of printer and may, for example, have about one hundred and fiftydrop dispensing orifices in each of two parallel rows, about sixchambers for holding a fluid for array fabrication, such as for examplepolynucleotide solution and the like, communicating with about threehundred ejectors which are positioned in the chambers opposite acorresponding orifice. Each orifice with its associated ejector andportion of the chamber, defines a corresponding pulse jet with theorifice acting as a nozzle. Thus, there are about three hundred pulsejets in the exemplary embodiment described above, although it will beappreciated that a given head system may, for example, have more or lesspulse jets as desired (for example at least about ten or at least aboutone hundred pulse jets). In this manner, application of a singleelectrical pulse to an ejector causes a droplet to be dispensed from acorresponding orifice.

The size of each orifice in the orifice plate may vary, where theorifice may have an exit diameter that ranges from about 1 μm to about 1mm, e.g., from about 5 μm to about 100 μm, e.g., from about 10 μm toabout 60 μm.

Each ejector is in the form of a piezoelectric ejector element (althoughan electrical resistor operating as a heating element may also be used),which is electrically connected to a source of electrical energy. Inaccordance with embodiments of the subject invention, the electricalenergy provided to each ejector is controllable, e.g., prior to eachejection of fluid, to deliver a suitable pulse of electricity toactivate the ejector on demand and eject a specific volume of fluid.

In certain embodiments of the foregoing exemplary configuration, abouttwenty orifices in each group of six reservoirs (many of the orificesmay be unused, e.g., plugged with a glue or the like), may be dispensingthe same fluid. Certain elements of each head may be adapted from partsof a commercially available inkjet print head device.

Exemplary head systems and other suitable dispensing head designs andfluid drop deposition apparatuses that may be employed in the practiceof the subject invention are described, e.g., in U.S. Pat. Nos.6,323,043 and 6,461,812, the disclosures of which are hereinincorporated by reference. However, other head system configurations maybe used.

As will be appreciated, the chambers may be filled with fluid using anysuitable technique, for example any suitable front- or back-loadingtechnique. For example, in certain embodiments, the chambers may befilled with fluid by contacting the exit ends of the orifices with aquantity of the fluid and then lowering the pressure upstream from theorifices by connecting a source of vacuum, resulting in drawing fluid inan upstream direction through the orifices into the chamber. Selecteddifferent fluids (or fluids containing different materials) may be drawninto the different chambers by contacting each orifice group (in fluidcommunication with a delivery chamber) with a different fluid. Othermethods of loading fluid into chambers may be employed as well.

The size of each orifice may vary, where the orifice may have an exitdiameter (or exit diagonal depending upon the particular format of thedevice) that ranges from about 1 μm to about 1 mm, e.g., from about 5 μmto about 100 μm, e.g., from about 10 μm to about 60 μm. The fluidcapacity of a given chamber may also vary and may range from about 0.1pL to about 1 mL or more, e.g., from about 1.0 pL to about 1 mL or more.

As noted above, embodiments include controlling the amount of fluidejected from each orifice by controlling one or more parameters of theelectrical energy supplied to an ejector.

It is to be understood that the above-described head configurations areexemplary only and various other dispensing head designs may be used.

Fabricating a Chemical Array Having Features of Different Sizes

As noted above, embodiments of the subject methods include controllingthe amount of fluid ejected from each orifice of a dispensing head,e.g., for each ejection of fluid (activation event) from each orifice,to control the size of each feature of a chemical array such as anucleic acid array, polypeptide array, etc. Accordingly, embodiments ofthe subject invention include modulating at least one parameter of afluid drop deposition device to selectively control the amount of fluidejected from each ejector of a fluid dispensing head, where theparameter may be set before and/or during the fabrication process, i.e.,the parameter may be changed during the fabrication process of an array.Any suitable parameter or combination of parameters may be modulatedaccording to the subject invention. In certain embodiments, at least oneof these parameters is the applied activation signal provided to eachejector of each deposition head employed in the fabrication of an array.As such, certain embodiments include selectively adjusting theactivation signal used for each ejector of a deposition device todispense droplets from corresponding orifices of particular sizes.Accordingly, embodiments of the subject methods include providing auniquely adjusted ejector activation signal each time the depositionhead is activated (fired). As noted above, the waveform of theactivation signal for each ejector may be modulated to provide auniquely adjusted waveform each time the deposition head is activated(fired).

In practicing the subject methods, a fluid drop deposition device isloaded with a volume of the fluid to be deposited on an array substratesurface. By “loaded” is meant that the fluid is at least introduced intoa chamber of the device. In certain embodiments, the subject devices areemployed to deposit fluids that include a biopolymer or a precursorthereof, an activator fluid, etc. In other words, the fluids of interestinclude ones that are used in the fabrication of an array and include,but are not limited to, a biopolymer or a biomonomer or precursorsthereof, activator, linking agent, and the like. Biopolymers aregenerally biomolecules (e.g., naturally occurring molecules found inliving organisms or synthetic mimetics/analogues thereof), wherebiomolecules of interest include polypeptides, polysaccharides, nucleicacids and the like, as well as derivatives thereof, where of particularinterest in many embodiments are nucleic acids, includingoligonucleotides and polynucleotides, e.g., cDNA, or polypeptides, e.g.,proteins or fragments thereof. Biopolymer precursors include activatedmonomers, e.g., activated amino acids and nucleotides, employed in stepwise fabrication protocols in which biopolymeric ligands are grown on asurface of a substrate, as is known in the art. The fluid may or may notbe aqueous, depending on the nature of the molecule to be delivered inthe fluid. For example, biopolymeric molecules may be delivered in anaqueous fluid, while activated monomers may require delivery in anon-aqueous fluid.

The fluid may be loaded into a given delivery chamber using anyconvenient method as noted above. Thus, conventional methods ofintroducing ink into inkjet heads may be employed. Where such methodsare employed, following loading of the fluid sample into the pulse jetdispensing head, it may be desirable to “prime” the device prior to use.One method of priming the device is to apply sufficient pressure to thefluid in the delivery chamber (or conversely negative pressure to theorifice) such that a volume of fluid is forced out of the orifice.

The following “front loading” method of loading fluid into the deliverychamber may be employed in certain embodiments, e.g., where minimalwaste of the fluid sample is desired (e.g. where the fluid is anexpensive or rare cDNA sample). In this method of fluid sample loading,the orifice is contacted with the fluid under conditions sufficient forfluid to flow through the orifice and into the chamber of the head,where fluid flow may be due, at least in part, to capillary forces incertain embodiments. To assist in the flow of fluid into the orifice,back pressure in the form of suction (i.e. negative pressure) may beapplied to the chamber of the head, where the back pressure may be atleast about 0.5, e.g., at least about 5, e.g., at least about 10 andeven as great as about 100 inches of H₂O or more. In general, eachchamber is subjected to the same back pressure. For a furtherdescription of this front loading procedure, see e.g., U.S. patentapplication Ser. No. 09/302,922; the disclosure of which is hereinincorporated by reference. As noted above, back loading methods ofloading fluid into a delivery chamber may also be employed.

The amount of fluid required to load the head is typically small, e.g.,may not exceed more than about 10 μl in certain embodiments, e.g., maynot exceed more than about 5 μl and in certain embodiments may notexceed more than about 2 μl. In certain embodiments, fluid may be loadedrapidly and efficiently into the chambers and reservoirs of thedeposition head assemblies from standard multiwell plates, e.g., 96 wellmicrotitre plates, 384 well microtitre plates, and the like.

Following loading of the dispensing head, e.g., by front or back loadingas described above or any other suitable method, the head is employed todeposit a quantity of at least one fluid onto the surface of asubstrate. In the broadest sense, the subject methods may be used todeposit a volume of fluid onto any structure, specifically a surface, ofany substrate, where the substrate may be a planar structure in certainembodiments.

To deposit fluid onto a substrate surface according to the subjectmethods, the loaded pulse jet head is positioned in opposingrelationship relative to the surface of the substrate (e.g. with an XYZtranslational means), where the orifice(s) is in opposition to theposition on the array surface at which deposition of the fluid isdesired. The distance between the orifice(s) and the substrate surfacewill not be so great that the volume of protein fluid cannot reach thesubstrate surface and produce a spot in a reproducible manner. As such,the distance between the orifice(s) and the substrate surface may rangefrom about 10 μm to about 10 mm, e.g., from about 100 μm to about 2 mm,e.g., from about 200 μm to about 1 mm.

Before, during or after the deposition head is placed into positionrelative to the substrate surface, a suitable waveform commensurate withthe amount of fluid to be dispensed from a corresponding orifice, isapplied to each ejector of the pulse jet head to actuate the ejector todispense a volume of fluid. For example, in many embodiments the ejectoris a piezoelectric ejector employing a piezoelectric crystal, e.g., alead-zirconate-titanate (“PZT”) material or the like, that changes shapeand/or vibrates when an electric field is applied across it.Accordingly, a voltage applied across a piezoelectric ejector causes itto modulate, e.g., grow in size, causing fluid to be ejected from theorifice. A feature of embodiments of the subject methods, described ingreater detail below, includes selectively modulating the voltageapplied across each piezoelectric element of a deposition head, which inturn controls the amount of fluid ejected from a corresponding orifice,to eject a specific volume of fluid from the orifice.

The subject methods are capable of depositing an extremely small volumeof fluid onto a substrate surface, the subject methods may be used todeposit a pico liter quantity of fluid onto a substrate in certainembodiments. By “pico liter quantity” is meant a volume of fluid that isat least about 0.1 pl, e.g., at least about 1 pl, e.g., at least about10 pl, where the volume may be as high as about 250 pl or higher, wherein certain embodiments the amount of fluid may not exceed about 100 nL,e.g., may not exceed about 1 μl. For example in certain embodiments theamount or volume of fluid that is forced out or expelled from the firingchamber may range from about 0.1 to 2000 pl, e.g., from about 0.5 to 500pl, e.g., from about 1.0 to 250 pl. The speed at which the fluid may beexpelled from the firing chamber may be at least about 1 m/s, e.g., atleast about 10 m/s and may be as great as about 20 m/s or greater.

Upon actuation of the pulse jet head of the subject invention, asdescribed above, fluid is expelled from the orifice and travels to thesubstrate surface. Upon contact with the substrate surface, thedeposited fluid typically forms a spot (feature) on the substratesurface. As mentioned above, by controlling certain parameters of thedispensing head such as the applied activation signal, e.g., thewaveform, applied to each ejector of the pulsejet head, the spotdimensions may be controlled and varied if desired such that spots ofvarious sizes may be produced. For example, the subject methods may beemployed to produce features having a characteristic length scale suchas features having widths (that is, diameter, for a round feature)ranging from about 10 μm to about 1.0 cm. In those embodiments wherevery small features are desired, small features that have acharacteristic length scale such as features having widths ranging fromabout 1.0 μm to 1.0 mm may be produced, e.g., from about 5.0 μm to 500μm, e.g., from about 10 μm to 200 μm. In certain embodiments, thefeatures may have a characteristic length scale such as features havingwidths ranging from about from about 30 to 100 μm.

As noted above, application of an electrical pulse to an ejector such asa piezoelectric ejector or the like causes a droplet of fluid to bedispensed from a corresponding orifice, where the size of the droplet isat least dependant in part on the particular electrical pulse applied tothe ejector. According to embodiments of the subject invention, theactivation signal applied to each ejector (which may be a single ejectoror a plurality of ejectors) of a deposition head is modulated to be setto a specific activation signal or adjusted to a particular activationsignal setting where the different ejectors of a dispensing head may usedifferent signals, but where each ejector is capable of beingselectively set to a respective activation signal, which setting isindependent of the signal set for any other ejector. The signalsemployed may differ in one or more respects such as waveform, and thelike. The activation signals, e.g., waveforms, may be set or adjustedfor each ejector one time prior to fabricating an array, or may be setor adjusted before each firing of the head, i.e., prior to eachactivation event of a pulse jet. In other words, embodiments includecontinually changing one or more activation signals, e.g., with respectto waveforms, provided to one or more ejectors of a deposition head,where such may be performed one or more times during an arrayfabrication procedure. Accordingly, the amount of fluid expelled fromdifferent pulse jets of the same deposition head may be the same or maybe different, where such is dependant at least in part on the activationsignal, e.g., the unique waveform, provided to the ejectors.

The exact activation signal parameters vary depending on the particulardeposition head, ejector, and the like. For piezoelectric ejectors,voltages may range from about 10 volts to about 150 volts, in certainembodiments.

The number of different waveforms per swath that may be utilized mayrange from about 2 to about several thousands or more, e.g., from about2 to about hundreds of thousands or more. In many embodiments, thewaveform is analogous to a step waveform such as a top-hat profilewaveform, as shown in FIG. 1.

For example, fluid may be loaded as described above, and before, duringor after the fluid is loaded (but in any event prior to an activationevent of the pulse jet), the waveforms and/or other signal parameter(but in any event a parameter that affects the volume dispensed from arespective orifice) for each ejector of a deposition head used in thefabrication (certain ejectors may not be used and instead may be masked)may be determined and set, where the waveform and/or other signalparameter provided to any given ejector may stay the same for allactivation events or may change for two or more activation events of anarray fabrication.

Accordingly, at some time prior to activation of each ejector of a pulsejet, a waveform and/or other signal parameter(s) suitable for theparticular droplet size (feature size) desired to be ejected from eachpulse jet of the deposition head used is determined. Suitable waveformsand/or one or more other signal parameter may be determined by employinga database of feature size and/or droplet size and correspondingwaveforms (and/or one or more other signal parameter). For example, thedrop deposition apparatus employed in the fabrication of a biopolymericarray may include (may be operatively coupled to) a waveform generator,such as a waveform circuit, that stores a plurality of waveform signalsfor a plurality of different, fluid volumes, and outputs the waveformsignals to the respective ejectors as needed.

Embodiments include employing an array layout. An “array layout” refersto one or more physical, chemical or biological characteristics of thearray, such as feature positioning, one or more feature dimensions suchas feature size, density, and the like, some indication of an identityor function (for example, chemical or biological) of a moiety at a givenlocation, how the array should be handled (for example, conditions underwhich the array is exposed to a sample, or array reading specificationsor controls following sample exposure), and the like. Othercharacteristics may include, but are not limited to, feature positioningon the substrate, one or more other feature dimensions such as probedensity, feature density, and the like, trigger tables, orifice maskingconfiguration (if any), ejector gain, indication of a moiety at a givenlocation, etc. In certain embodiments the array layout is provided as adata file, e.g., which may be in the form of text, such as an XML fileor the like which may be automatically communicated to a fluiddepositions apparatus.

For example, certain embodiments may be illustrated by the block diagramof FIG. 2. Embodiments of the subject invention may include inputting600 or otherwise selecting data that includes parameters used tofabricate an array, and which may at least includes the desired featuresizes of the array to be fabricated. The data may be in any suitableform and may be communicated to a suitable manufacturing executionsystem (“MES”) 610 or the like as is known in the art which is may begenerally described as a software/hardware based manufacturing systemthat collects and organizes data used in manufacturing processes. TheMES, if employed, may include a database 620 of array feature processparameters, including a population of various feature sizes, featurecomposition, etc. Such may also include various patterns of features,where the patterns may include, e.g., organized rows and columns offeatures, e.g. a grid of features, across the substrate surface, aseries of curvilinear rows across the substrate surface, e.g. a seriesof circles such as a series of concentric circles or semi-circles offeatures, and the like.

The particular features sizes for the array to be fabricated may be, butare not necessarily, selected from this or other analogous database incertain embodiments. For example, a user may input certain arrayrequirements, general or specific, and the database may be employed toassist in the selection the specific array design parameters that wouldachieve the inputted requirements, e.g., by employing computing means.XML file generation software 630 or other analogous software (or othersuitable method), may provide an output, e.g., in the form of one ormore data files such as one or more XML files 650 or the like, of theparticular array design parameters, one of which being feature size ofthe features of the array. The particular activation signal for eachejector may then be determined, and adjusted if needed, to accomplishthe particular droplet size desired to be dispensed from a respectiveorifice (and thus fabricated feature size), e.g., by employing suitablecomputing means. For example, the determined feature sizes of the arraymay then be communicated, manually or automatically, to a fluid dropdeposition device 660 and particularly to suitable deposition headcontrol hardware 670, manually or automatically, and the activationsignal for each ejector may be adjusted 680, manually or automatically,to provide the desired activation signal to each ejector of thedeposition head to provide droplet size commensurate with the desiredfeature size (or not if the waveform that is currently set is thedesired waveform). In certain embodiments, the relevant array layoutinformation, e.g., present as one or more data files (e.g., one or moreXML files or the like), may first be parsed by a suitable file parser665 or the like.

In certain embodiments, sizes of the features of an array may be chosenwith respect to the at least suspected abundance of a target in a samplefor which the array is designed to be used. For example, if a target isat least suspected of being present in such a sample in low or very lowabundance amounts, features to which the target sample is to becontacted may be fabricated to be smaller in size with respect to atleast one dimension thereof such as width or the like relative totargets having an average, high or very high abundance of targets, andvice versa.

Activation signals may be selectively set for each ejector to providefeatures having characteristic length scales, e.g., features havingwidths (that is, diameter, for a round spot) in the range from about 10μm to about 1.0 cm. In certain embodiments, features may have widths inthe range from about 1.0 μm to about 1.0 mm, e.g., from about 5.0 μm toabout 500 μm, e.g., from about 10 μm to about 200 μm, e.g., from about50 μm to about 150 μm. Non-round features may have area rangesequivalent to that of circular features with the foregoing width(diameter) ranges. Accordingly, in certain embodiments, the activationsignal provided to each ejector for each activation event may be thesame, thus dispensing the same size droplets from the correspondingorifices to provide features of the same sizes. In certain otherembodiments, activation signals provided to at least two ejectors may bedifferent, thus dispensing the different sizes droplets from thecorresponding orifices to provide features of different sizes. Incertain embodiments, methods include determining an at least suspected,including unknown, abundance of target in a sample to which the array isdesigned to be used and determining feature size (and/or droplet size)therefrom, where such may be accomplished manually or automatically froma suitable database of target abundance amounts and correspondingfeature sizes (and/or droplet sizes).

In certain embodiments it may be desirable to fabricate an array offeatures to low or very low abundance targets in a sample. Accordingly,it may be advantageous to fabricate these features of larger sizesrelative to features directed to average, high or very high abundancetargets. Accordingly, the waveforms provided to the ejectors of thefeatures to low or very low abundance targets in a sample are suitablyset to dispense larger amounts of fluid to provide these relativelylarger feature sizes (and vice versa). For example, where the amount oftarget in or suspected of being in a sample is a low or very lowabundant target, the waveforms provided to the appropriate ejectors areselected to provide a fluid droplet commensurate with providing a roundfeature to such a target that may have dimensions corresponding to thisabundance level, e.g., may have a characteristic length scale (e.g., mayhave a width or the like) that ranges from about 100 μm to about 1 mm.In certain embodiments, features to high or very high abundance targetsmay have smaller sizes relative to features for average, low or very lowabundance targets. For example, where the amount of target in orsuspected of being in a high or very high abundant target, the waveformsprovided to the appropriate ejectors are selected to provide a fluiddroplet commensurate with providing a round feature to such a targetthat may have dimensions corresponding to this abundance level, e.g.,may have a characteristic length scale (e.g., may have a width or thelike) that ranges from about 5 μm to about 100 μm.

Arrays fabricated in accordance with the subject invention typicallyinclude at least two distinct polymers (i.e., two distinct probes),e.g., that differ by monomeric sequence, attached to different and knownlocations on the array substrate surface. As noted above, each distinctpolymeric sequence of the array is typically present as a composition ofmultiple copies of the polymer on a substrate surface, e.g., as a spotor feature on the surface of the substrate. In accordance with thesubject invention, the size of each feature may be precisely andindependently controlled such that features need not all be of the samesize and some or all of the features may be of different sizes. Forexample, in certain embodiments, features of an array that differ inmonomeric sequences (i.e., differ in composition) may be of differentsizes. Embodiments may also include features of an array that includethe same monomeric sequences or are of the same composition (i.e.,replicate features) may be of different sizes.

Any given substrate may carry one, two, four or more arrays disposed ona surface of the substrate. Depending upon the use, any or all of thearrays may be the same or different from one another and each maycontain multiple spots or features as noted above. For example, aplurality of arrays may be stably associated with one substrate, wherethe arrays are spatially separated from some or all of the other arraysassociated with the substrate. In certain embodiments, a substrate maycarry at least two arrays. The at least two arrays may include some orall of the features of the same composition, but some or all of whichdiffer in feature size.

The probes may be immobilized on surfaces of any of a variety ofdifferent substrates, including both flexible and rigid substrates.Typically, the materials provide physical support for the depositedmaterial and endure the conditions of the deposition process and of anysubsequent treatment or handling or processing that may be encounteredin the use of the particular array. The array substrate may take any ofa variety of configurations ranging from simple to complex. Thus, thesubstrate could have generally planar-form, as for example, a slide orplate configuration, such as a rectangular or square disc. In manyembodiments, the substrate will be shaped generally as a rectangularsolid, having a length in the range of about 4 mm to 200 mm, usuallyabout 4 mm to 150 mm, more usually about 4 mm to 125 mm; a width in therange of about 4 mm to 200 mm, usually about 4 mm to 120 mm, and moreusually about 4 mm to about 80 mm; and a thickness in the range of about0.01 mm to about 5 mm, usually from about 0.1 mm to about 2 mm and moreusually from about 0.2 mm to about 1 mm. However, larger or smallersubstrates may be and can be used. Substrates of other configurationsand equivalent areas may be employed. The configuration of the array maybe selected according to manufacturing, handling, and useconsiderations.

The substrates may be fabricated from any of a variety of materials. Incertain embodiments, such as for example where production of bindingpair arrays for use in research and related applications is desired, thematerials from which the substrate may be fabricated should ideallyexhibit a low level of non-specific binding during hybridization events.In many situations, it will also be preferable to employ a material thatis transparent to visible and/or UV light. For flexible substrates,materials of interest include: nylon, both modified and unmodified,nitrocellulose, polypropylene, and the like, where a nylon membrane, aswell as derivatives thereof, may be particularly useful in thisembodiment. For rigid substrates, specific materials of interestinclude: glass; fuse silica; silicon, plastics (for examplepolytetraflouroethylene, polypropylene, polystyrene, polycarbonate, andblends thereof, and the like); metals (for example, gold, platinum, andthe like).

The substrate surface onto which the probes are immobilized may besmooth or substantially planar, or have irregularities, such asdepressions or elevations. The substrate surface may be modified withone or more different layers of compounds that serve to modify theproperties of the surface in a desirable manner. Such modificationlayers of interest include: inorganic and organic layers such as metals,metal oxides, polymers, small organic molecules and the like. Polymericlayers of interest include layers of: peptides, proteins, polynucleicacids or mimetics thereof (for example, peptide nucleic acids and thelike); polysaccharides, phospholipids, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyetheyleneamines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, and the like, wherethe polymers may be hetero- or homopolymeric, and may or may not haveseparate functional moieties attached thereto (for example, conjugated).

The fabrication of the arrays of the subject invention may be partiallyor completely automated. For example, an automated system as illustratedin FIG. 3 may be employed. At some point prior to fabrication, anactivation signal for each ejector employed in the fabrication procedureis determined and communicated to suitable activation generatinghardware/software so that a unique activation signal is applied to eachejector and a suitable volume of fluid, commensurate with a desiredfeatures size, is dispensed from each pulse jet used. For example, asnoted above, such automated apparatuses may include a waveform generatorsuch as a waveform circuit or the like, that stores a plurality ofwaveform signals, each stored waveform signal corresponding to adifferent amount of fluid volume ejected from an orifice. The waveformcircuit may output waveform signals to appropriate fluid drop depositionapparatus hardware so that a particular waveform signal may be appliedto a particular ejector of a deposition head, commensurate with theamount of fluid desired to be dispensed from a respective orifice for agiven activation event. To accomplish this, the same or differentcircuit may be provided for generating signals based on the outputwaveform signals from the waveform circuit and for outputting thesignals to an ejector.

The above methods may be substantially, if not completely automated, sothat fluid may be loaded and deposited onto a surface automatically. Assuch, the subject methods are amenable to high throughput applications,e.g., high throughput manufacturing applications. In automated versionsof the subject methods, automated apparatuses are employed and typicallyinclude at least a manner for precisely controlling the position of oneor more dispensing heads with respect to a substrate surface (an XYZtranslational mechanism) and for firing the head. Such automated devicesare well known to those of skill in the art and are disclosed in U.S.Pat. Nos. 6,242,266; 6,232,072; and 6,180,351; as well as in copending,commonly assigned U.S. Patent Serial No. application Ser. No.10/281,408, the disclosures of which are herein incorporated byreference.

One such automated system that may be employed in the practice of thesubject methods is described with reference FIG. 3, which shows anapparatus capable of executing a method of the present invention. Whilethe apparatus is described as configured for use with a large substrate19 which will later be cut into individual substrates 10 to providearray assemblies 15 that include a substrate 10 and at least one arraythereon it will be apparent that the apparatus may also be employed tofabricate one or more arrays on a substrate that is not later cut, butis used as fabricated. The apparatus shown essentially has two sections,a first, optional section on which a surface 11 a of the substrate 19may be functionalized if desired, and a second section in which thearray is fabricated on a surface of the substrate 19 such as afunctionalized surface (if performed). While these two sections areshown as part of one apparatus in FIG. 3, it will be appreciated thatthey may be entirely separate with the first section preparing manyfunctionalized substrates 19 which are forwarded to the fabricationsection for array fabrication, with their possibly being one or morefirst sections and one or more second sections remote from each other.

The first, optional section of the apparatus of FIG. 3 includes a firstsubstrate station 70 which can retain a mounted substrate 19, a thirdtransporter 70, a deposition head retainer 76, and a first dropdeposition system in the form of a pulse jet head 78 system. Pulse jethead system 78 may be analogous to that described above and includeabout one, about two, about three or more (e.g., about ten or more)pulse jet heads which deliver drops of fluid onto surface 11 a ofsubstrate 19 all so as to functionalize that surface. Drops may bedelivered from head 78 while substrate 19 is advanced beneath it bytransporter 70, all under control of a processor 140.

The second section of the apparatus of FIG. 3 includes substrate station20 (sometimes referenced as a “substrate holder”) on which a substrate19 may be mounted and retained. Pins or similar means (not shown) may beprovided on substrate station 20 by which to approximately alignsubstrate 19 to a nominal position thereon (with optional alignmentmarks 18 on substrate 19 being used for more refined alignment).Substrate station 20 may include a vacuum chuck connected to a suitablevacuum source (not shown) to retain a substrate 19 without exerting toomuch pressure thereon, since substrate 19 may be made of glass. Anoptional flood station 68 may be provided which can expose the entiresurface of substrate 19, when positioned at station 68 as illustrated inbroken lines in FIG. 3, to a fluid typically used in the in situprocess, and to which all features must be exposed during each cycle(for example, oxidizer, deprotection agent, and wash buffer). In thecase of deposition of a previously obtained polynucleotide, floodstation 68 need not be present.

A second drop deposition system is present in the form of a dispensinghead 210 which is retained by a head retainer 208. As mentioned abovethough, the head system may include more than one head 210 retained bythe same head retainer 208 so that such retained heads move in unisontogether. The transporter system may include a carriage 62 connected toa first transporter 60 controlled by processor 140 through line 66, anda second transporter 100 controlled by processor 140 through line 106.Transporter 60 and carriage 62 are used to execute one axis positioningof station 20 (and hence mounted substrate 19) facing the dispensinghead 210, by moving it in the direction of axis 63, while transporter100 is used to provide adjustment of the position of head retainer 208(and hence head 210) in a direction of axis 204 (and therefore move head210 in the direction of travel 204 a which is one direction on axis204). In this manner, head 210 can be scanned line by line alongparallel lines in a raster fashion, by scanning along a line oversubstrate 19 in the direction of axis 204 using transporter 100, whileline to line transitioning movement of substrate 19 in a direction ofaxis 63 is provided by transporter 60. Transporter 60 may also movesubstrate holder 20 to position substrate 19 in flood station 68 (asillustrated by the substrate 19 shown in broken lines in FIG. 3). Head210 may also optionally be moved in a vertical direction 202, by anothersuitable transporter (not shown) and its angle of rotation with respectto head 210 also adjusted. It will be appreciated that other scanningconfigurations could be used during array fabrication. It will also beappreciated that both transporters 60 and 100, or either one of them,with suitable construction, could be used to perform the foregoingscanning of head 210 with respect to substrate 19. Thus, when thepresent application recites “positioning”, “moving”, or similar, oneelement (such as head 210) in relation to another element (such as oneof the stations 20 or substrate 19) it will be understood that anyrequired moving can be accomplished by moving either element or acombination of both of them. The head 210, the transporter system, andprocessor 140 together act as the deposition system of the apparatus. Anencoder 30 communicates with processor 140 to provide data on the exactlocation of substrate station 20 (and hence substrate 19 if positionedcorrectly on substrate station 20), while encoder 34 provides data onthe exact location of holder 208 (and hence head 210 if positionedcorrectly on holder 208). Any suitable encoder, such as an opticalencoder, may be used which provides data on linear position.

Processor 140 may also have access through a communication module 144 toa communication channel 180 to communicate with a remote station.Communication channel 180 may, for example, be a Wide Area Network(“WAN”), telephone network, satellite network, or any other suitablecommunication channel. Array parameters such as certain waveforms may becommunicated to the processor via such a remote station.

Each of one or more heads 210 may be of a type described above, and may,for example, include five or more dispensing chambers (e.g., at leastone for each of four nucleoside phosphoramidite monomers plus at leastone for an activator solution) each communicating with a correspondingset of multiple drop dispensing orifices and multiple ejectors which arepositioned in the chambers opposite respective orifices. As describedabove, application of an electric-pulse to an ejector will cause adroplet to be dispensed from a corresponding orifice, its sizedetermined at least in part by the magnitude of the applied electricpulse. Each ejector, e.g., piezoelectric ejector, is under the controlof processor 140, which processor, under the control of a suitablesoftware program, adjusts the activation signal provided to eachejector, commensurate with a particular desired droplet size to beejected. Processor 140 may communicate with memory 141 which may includea database of various droplet sizes and/or feature sizes, eachcorresponding to a particular waveform. Accordingly a suitable waveformfor each ejector may be selected and the processor may perform all ofthe steps necessary to provide the suitable waveform to each ejector.Multiple heads could be used instead of a single head 210, each beingsimilar in construction to head 210 and being movable in unison by thesame transporter or being provided with respective transporters undercontrol of processor 140 for independent movement. In this alternateconfiguration, each head may dispense a corresponding biomonomer (forexample, one of four nucleoside phosphoramidites) or an activatorsolution.

The apparatus further includes a display 310, speaker 314, and operatorinput device 312. Operator input device 312 may, for example, be akeyboard, mouse, or the like. Input system may be, or may be operativelycoupled to, an MES system or the like as described above, and/or a datafile (e.g., which may be in the form of text, such as an XML file or thelike), or any analogous component that includes and is capable ofcommunicating array parameters such as desired feature sizes to theprocessor. Processor may be capable of receiving array layoutinformation, e.g., in the form of one or more data files (e.g., whichmay be in the form of text such as such as one or more XML files or thelike), from input device 312, and parsing the parameters (see forexample FIG. 2), to at least provide desired feature sizes, which datamay then be used to execute the step necessary to determine and providesuitable waveforms to each ejector.

Processor 140 has access to a memory 141, and controls print head system78 and print head 210 (specifically, the activation of the ejectorstherein), operation of the transporter system and the third transporter72, and operation of display 310 and speaker 314. Memory 141 may be anysuitable device in which processor 140 can store and retrieve data, suchas magnetic, optical, or solid state storage devices (including magneticor optical disks or tape or RAM, or any other suitable device, eitherfixed or portable). Processor 140 may include a general purpose digitalmicroprocessor suitably programmed from a computer readable mediumcarrying necessary program code, to execute all of the steps required bythe present invention, or any hardware or software combination whichwill perform those or equivalent steps. The programming may be providedremotely to processor 141 through communication channel 180, orpreviously saved in a computer program product such as memory 141 orsome other portable or fixed computer readable storage medium using anyof those devices mentioned below in connection with memory 141. Forexample, a magnetic or optical disk 324 a may carry the programming, andcan be read by disk writer/reader 326. A cutter 152 may be provided tocut substrate 19 into individual array assemblies 15.

As shown in FIG. 3, substrate 19 is already mounted on substrate station70. If it is desired to surface modify or functionalize substrate 19,such may be accomplished at an optional fuctionalization station oroptional first fabrication station 70. Station 70 may be used to providea linking layer on the substrate surface (see for example U.S.application Ser. No. 10/281,408, the disclosure of which is hereinincorporated by reference). Station 70, may employ transporter system 72to advance the mounted substrate 19 beneath head system 78 while dropsof fluid, e.g., for a linking layer, are deposited on the substrate. Forexample, in fabricating arrays by depositing previously obtainedbiopolymers or by the in situ method, the entire region on the substratesurface on which an array will be formed (an “array region”) may beexposed to one or more reagents. For example, in either method, arrayregions may be exposed to one or more linker compositions to form asuitable linker layer on the surface which binds to both the substrateand biopolymer or biomonomer. Particularly useful linker compositionsand methods are disclosed in U.S. Pat. Nos. 6,319,674 and 6,444,268which may use various silane based compounds as linkers or other surfacemodifying agents (for example, to modify the surface energy to controldeposited drop spread).

The substrate 19 with the modified surface such as a linking layersurface (if performed) may then be transferred to the substrate station20 either manually or by the robot arm, as which station one or morearrays will be fabricated on the substrate surface 11 a. In thissequence it will be assumed that processor 140 is already programmedwith the necessary layout information, which at least includes thedesired feature sizes, to fabricate at least one array on the substratesurface (Alternatively, characteristics of a particular sample to beused with the fabricated array(s) may be provided to the processor,which may then perform the steps necessary to use this information todetermine suitable feature sizes (e.g., by accessing a database ofmemory 141), e.g., based on target abundance, and thus suitablewaveforms to accomplish the appropriate feature sizes).

Using information such as the foregoing array layout and the number andlocation of pulse jets in head 210, processor 140 may then determine areagent drop deposition pattern and adjust the activation signal foreach ejector of head 210 accordingly to provide features of sizescommensurate with that of the pattern. Alternatively, such a pattern maybe determined by another processor (such as a remote processor) andcommunicated to memory 141 through communication channel 180 or byforwarding a portable storage medium carrying such pattern data forreading by reader/writer 326. Processor 140 controls fabrication, inaccordance with the deposition pattern, to generate the one or morearrays on sections of substrate 19 which may later be cut intoindividual substrates 10 to provide a plurality of array assemblies 15.

Depending on the particular activation signal provided to an ejector,drops of fluid of particular sizes used in the fabrication of the one ormore arrays are deposited from the head while moving along each line ofthe raster during scanning. For example, different waveforms provided todifferent ejectors will result in different droplet sizes beingdispensed from the orifices of ejectors with different waveformsapplied. No drops are dispensed for features or otherwise during linetransitioning. Processor 140 also sends substrate 19 to optional floodstation 68 for cycle intervening or final steps as required, all inaccordance with the conventional in situ array, e.g., polynucleotidearray fabrication process, described above. As a result of the above, atleast one array is fabricated on substrate 19, where an array may havefeatures of different sizes.

The substrate 19 may then be sent to a cutter 152 wherein sections ofsubstrate 19 are separated into multiple substrates 10 carrying one ormore arrays, to provide multiple array assemblies 15. One or more arrayassemblies 15 may then be placed in a package 340 and forwarded to oneor more remote users to be used in an array assay.

During array fabrication errors may be monitored and used in any of themanners described in U.S. Patent Application “Polynucleotide ArrayFabrication” by Caren et al., Ser. No. 09/302,898 filed Apr. 30, 1999,and U.S. Pat. No. 6,232,072, the disclosures of which are hereinincorporated by reference. Also, one or more identifiers in the form ofbar codes may be attached or printed onto sections of substrate 19,e.g., defining particular parameters of the arrays such as the featuresizes and addresses of such. Such may be attached to the substrate,e.g., before entering, or after leaving, optional station 70, or beforeentering or after leaving the fabrication station 20. If bar codes arepresent before entering one of the fabrication stations, they mayinclude an indication of the location of different regions on asubstrate to which probes or probe precursors are to be fabricated atdifferent feature sizes. They can then be read by a bar code reader (notshown) at a fabrication station, and received by processor 140 to thencontrol the drop deposition system to form the one or more arrays withfeatures of different sizes either within the same or different arrays,e.g., features of the same or different probe composition in one of theregions which are repeated in another of the regions at differentfeature sizes. Any of the foregoing types of information on thedifferent regions can be contained within the bar codes 356 (or otheridentifiers) or in a file previously linked to them. Regardless of theforegoing, at any point in the operation of the apparatus of FIG. 3,processor 140 may associate each array with an identifier such as a barcode, which identifier may carry an indication of the different featuresizes of the same or different probe composition or may be linked to afile carrying such information. The file and linkage may be stored byprocessor 140 and saved into memory 141 or may be written onto aportable storage medium 324 b which is then placed in the same package340 as the corresponding array assembly 15 for shipping to a remotecustomer. The actual indication may take many forms. For example, one ormore of the bar codes associated with the arrays on the same substrate10 may specify that one array is the same as another array such asarray, but that the region carrying one has a feature size which is aproportion of the region at the other. Alternatively, absolute featuresizes may be provided for each array in its associated bar code.

Optionally other characteristics of the fabricated arrays may beincluded in the code applied to the array substrate or a housing, or afile linkable to such code, in a manner as described in U.S. Pat. No.6,180,351, incorporated herein by reference.

It will be understood that there may be multiple user stations, eachremote from the fabrication station and each other, in which case thefabrication station acts as a central fabrication station (that is, afabrication station which services more than one remote user station atthe same or different times). One or more such user stations may be incommunication with the fabrication station at any given time. It willalso be appreciated that processors 140 and 162 may be programmed fromany computer readable medium carrying a suitable computer program. Forexample, such a medium can be any memory device such as those describedin connection with memory 141, and may be read locally (such as byreader/writer 326 in the case of processor 140 or writer/reader 186 inthe case of processor 162) or from a remote location throughcommunication channel 180.

A variety of different chemical arrays may be produced according to thesubject methods including biopolymeric arrays such as nucleic acidarrays, peptide arrays, and the like.

Chemical Arrays Having Features of Different Sizes

Also provided by the subject invention are chemical arrays of nucleicacids (e.g., oligonucleotides, polynucleotides), peptides (e.g.,polypeptides, proteins, antibodies) or other molecules capable ofbinding with target biomolecules in a solution (e.g., nucleic acids,proteins, etc.), which arrays may have features of different sizes,e.g., customized or tailored such as for example with respect to the atleast anticipated abundance of a target in a sample for which the arrayis designed to assay. That is, an array of probes (i.e., binding agentsor members of a binding pair in this context) covalently bonded to asubstrate surface in the form of an “array” or pattern is provided wherecertain parameters of the array have been tailored to the amount of aparticular target present or suspected of being present in a sample forwhich the array is designed to assay. Such arrays find use in a varietyof different fields, e.g., genomics (in sequencing by hybridization, SNPdetection, differential gene expression analysis, identification ofnovel genes, gene mapping, finger printing, mutations analysis, etc.),proteomics, and the like.

The subject arrays typically include at least two distinct polymers thatdiffer by monomeric sequence attached to different and known locationson the substrate surface. Each distinct polymeric sequence of the arraymay be present as a composition of multiple copies of the polymer on asubstrate surface, e.g., as a spot or feature on the surface of thesubstrate, where the size of the features of an array may be the same ormay be different. The number of distinct polymeric sequences, and hencespots or similar structures, present on the array may vary, where atypical array may contain more than about ten, more than about onehundred, more than about one thousand, more than about ten thousand oreven more than about one hundred thousand features in an area of lessthan about 20 cm² or even less than about 10 cm². For example, featuresmay have characteristic length scales, e.g., features may have widths(that is, diameter, for a round spot), in the range from about 10 μm toabout 1.0 cm. In other embodiments, each feature may have acharacteristic length scale such as a width in the range from about 1.0μm to about 1.0 mm, e.g., from about 5.0 μm to about 500 μm, e.g., fromabout 10 μm to about 200 μm, e.g., from about 50 μm to about 150 μm.Non-round features may have area ranges equivalent to that of circularfeatures with the foregoing width (diameter) ranges. At least some, orall, of the features are of different compositions (for example, whenany repeats of each feature composition are excluded, the remainingfeatures may account for at least about 5%, 10% or 20% of the totalnumber of features). Interfeature areas will typically (but notessentially) be present which do not carry any polynucleotide (or otherbiopolymer or chemical moiety of a type of which the features arecomposed). The spots or features of distinct polymers present on thearray surface are generally present as a pattern, where the pattern maybe in the form of organized rows and columns of spots, e.g. a grid ofspots, across the substrate surface, a series of curvilinear rows acrossthe substrate surface, e.g. a series of concentric circles orsemi-circles of spots, and the like.

In certain embodiments the chemical arrays are arrays of polymeric orbiopolymeric ligands or molecules, i.e., binding agents. In manyembodiments of interest, the arrays are arrays of nucleic acids,including oligonucleotides, polynucleotides, cDNAs, mRNAs, syntheticmimetics thereof, and the like.

Each array may cover an area of less than about 100 cm², or even lessthan about 50 cm², 10 cm² or 1 cm². In many embodiments, the substratecarrying the one or more arrays will be shaped generally as arectangular solid (although other shapes are possible), having a lengthof more than about 4 mm and less than about 1 m, usually more than about4 mm and less than about 600 mm, more usually less than about 400 mm; awidth of more than about 4 mm and less than about 1 m, usually less thanabout 500 mm and more usually less than about 400 mm; and a thickness ofmore than about 0.01 mm and less than about 5.0 mm, usually more thanabout 0.1 mm and less than about 2 mm and more usually more than about0.2 and less than about 1 mm. With arrays that are read by detectingfluorescence, the substrate may be of a material that emits lowfluorescence upon illumination with the excitation light. Additionallyin this situation, the substrate may be relatively transparent to reducethe absorption of the incident illuminating laser light and subsequentheating if the focused laser beam travels too slowly over a region. Forexample, the substrate may transmit at least about 20%, or about 50% (oreven at least about 70%, 90%, or 95%), of the illuminating lightincident on the substrate as may be measured across the entireintegrated spectrum of such illuminating light or alternatively at 532nm or 633 nm.

FIGS. 6, 7 and 8, show chemical arrays of the present invention. FIG. 5shows array assembly 15 that includes a contiguous planar substrate 10carrying an array 112 disposed on a surface 11 a of substrate 10. Itwill be appreciated though, that more than one array (any of which arethe same or different) may be present on surface 11 a, with or withoutspacing between such arrays. That is, any given substrate may carry one,two, four or more arrays disposed on a front surface of the substrateand depending on the use of the array, any or all of the arrays may bethe same or different from one another and each may contain multiplespots or features where some or all of the features may be of differentsizes. The one or more arrays 112 usually cover only a portion of thesurface 11 a, with regions of the surface 11 a adjacent the opposedsides 113 c, 113 d and leading end 113 a and trailing end 113 b ofsubstrate 10, not being covered by any array 112. A second surface 11 bof the substrate 10 does not carry any arrays 112. Each array 112 may bedesigned for testing against any type of sample, whether a trial sample,reference sample, a combination of them, or a known mixture ofbiopolymers such as polynucleotides. Substrate 10 may be of any shape,as mentioned above.

Chemical array 112 may contain multiple spots or features 116 ofbiopolymers, e.g., in the form of polynucleotides. All of the features116 may be different, or some or all could be the same, but in any eventthe feature size of each feature has been precisely controlled asdescribed above, e.g., prior to each activation event. The interfeatureareas 117 could be of various sizes and configurations. Each featurecarries a predetermined biopolymer such as a predeterminedpolynucleotide (which includes the possibility of mixtures ofpolynucleotides).

Substrate 10 may carry on surface 11 a (and/or surface 11 b), anidentification code analogous to that describe above, e.g., in the formof bar code 356 or the like printed on the substrate in the form of apaper or plastic or electric label attached by adhesive or anyconvenient means. Identifiers such as other optical or magneticidentifiers could be used instead of bar codes which will carry theinformation discussed above. The identification code may containinformation relating to array 112, where such information may include,but is not limited to, an identification of array 112, i.e., layoutinformation relating to the array(s), including the feature sizes andaddresses thereof, etc. Each identifier may be associated with itscorresponding array by being positioned adjacent that array. However,this need not be the case and identifiers such as bar codes may bepositioned elsewhere on an array substrate, e.g., if some other means ofassociating each bar code with its corresponding array is provided (forexample, by relative physical locations). Further, a single identifiermight be provided which is associated with more than one array on a samesubstrate and such one or more identifiers may be positioned on aleading or trailing end of substrate. The substrate may further have oneor more fiducial marks for alignment purposes during array fabrication.

FIG. 6 shows an enlarged view of a portion of array 112 of FIG. 5showing features of differing sizes. As shown, array 112 includes afirst row R1 of features 116 a of a first size, a second row R2 offeatures 116 b of a second size, a third row R3 of features 116 c of athird size, a fourth row R4 of features 116 d of a fourth size and afifth row R5 of features 116 e of a fifth size. The compositions of thefeatures may all be the same or some or all of the features may be ofdifferent compositions (i.e., be different probes). For example, a givenrow may include features of different compositions, and other rows ofthe array may be replicates of those different composition features, butwhich differ in size. For example, first row R1 may include features ofdifferent compositions, which features are repeated in at least oneother row in a size that differs from the size of the features in firstrow R1, e.g., repeated in row R2 (or R2 . . . ). While the features ofarray 112 are shown arranged in rows/columns, it is to be understoodthat is configuration is for exemplary purposes only and is in no wayintended to limit the scope of the invention as the features of an arraymay be arranged in any suitable configuration.

FIGS. 7-8 show various exemplary arrays 112 a and 112 b, respectively,that may be provided by the subject invention. While arrays 112 a and112 b are shown on different substrates, however they may be on the samesubstrate which may or may not be later cut to individual arrayassemblies.

FIG. 7 shows a portion of an array assembly 515 having array 112 a offeatures 116 on substrate 10 a wherein the portion of array 112 a shownis configured generally as three columns C1, C2 and C3 of features, eachcolumn having features 116 h, 116 g and 116 f, respectively, that differin size from those of any other column. The probe composition in thefeatures may be the same or some may vary. For example, features acrossthe same row may have the same feature composition, but may differ infeature size and the compositions may differ amongst features of thesame column.

FIG. 8 shows array assembly 525 having array 112 b of features 116 i-116q on substrate 10 b configured generally as a circular array whereinsome or all of the features 116 may differ in size and/or composition.

A feature of the subject arrays, which feature results from the protocolemployed to manufacture the arrays, is that some or all of the featuresmay differ in size.

Utility

The subject arrays find use in a variety of different applications,where such applications are generally analyte detection applications inwhich the presence of a particular analyte (i.e., target) in a givensample is detected at least qualitatively, if not quantitatively.Protocols for carrying out such assays are well known to those of skillin the art and need not be described in great detail here. Generally,the sample suspected of containing the analyte of interest is contactedwith an array produced according to the subject methods under conditionssufficient for the analyte to bind to its respective binding pair member(i.e., probe) that is present on the array. Thus, if the analyte ofinterest is present in the sample, it binds to the array at the site ofits complementary binding member and a complex is formed on the arraysurface. The presence of this binding complex on the array surface isthen detected, e.g. through use of a signal production system, e.g. anisotopic or fluorescent label present on the analyte, etc. The presenceof the analyte in the sample is then deduced from the detection ofbinding complexes on the substrate surface. Specific analyte detectionapplications of interest include, but are not limited to, hybridizationassays in which nucleic acid arrays are employed.

In these assays, a sample to be contacted with an array may first beprepared, where preparation may include labeling of the targets with adetectable label, e.g. a member of signal producing system. Generally,such detectable labels include, but are not limited to, radioactiveisotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols,ligands (e.g., biotin or haptens) and the like. Thus, at some time priorto the detection step, described below, any target analyte present inthe initial sample contacted with the array may be labeled with adetectable label. Labeling can occur either prior to or followingcontact with the array. In other words, the analyte, e.g., nucleicacids, present in the fluid sample contacted with the array may belabeled prior to or after contact, e.g., hybridization, with the array.In some embodiments of the subject methods, the sample analytes e.g.,nucleic acids, are directly labeled with a detectable label, wherein thelabel may be covalently or non-covalently attached to the nucleic acidsof the sample. For example, in the case of nucleic acids, the nucleicacids, including the target nucleotide sequence, may be labeled withbiotin, exposed to hybridization conditions, wherein the labeled targetnucleotide sequence binds to an avidin-label or an avidin-generatingspecies. In an alternative embodiment, the target analyte such as thetarget nucleotide sequence is indirectly labeled with a detectablelabel, wherein the label may be covalently or non-covalently attached tothe target nucleotide sequence. For example, the label may benon-covalently attached to a linker group, which in turn is (i)covalently attached to the target nucleotide sequence, or (ii) comprisesa sequence which is complementary to the target nucleotide sequence. Inanother example, the probes may be extended, after hybridization, usingchain-extension technology or sandwich-assay technology to generate adetectable signal (see, e.g., U.S. Pat. No. 5,200,314).

In certain embodiments, the label is a fluorescent compound, i.e.,capable of emitting radiation (visible or invisible) upon stimulation byradiation of a wavelength different from that of the emitted radiation,or through other manners of excitation, e.g. chemical or non-radiativeenergy transfer. The label may be a fluorescent dye. Usually, a targetwith a fluorescent label includes a fluorescent group covalentlyattached to a nucleic acid molecule capable of binding specifically tothe complementary probe nucleotide sequence.

Following sample preparation (labeling, pre-amplification, etc.), thesample may be introduced to the array using any convenient protocol,e.g., sample may be introduced using a pipette, syringe or any othersuitable introduction protocol. The sample is contacted with the arrayunder appropriate conditions to form binding complexes on the surface ofthe substrate by the interaction of the surface-bound probe molecule andthe complementary target molecule in the sample. The presence oftarget/probe complexes, e.g., hybridized complexes, may then bedetected. In the case of hybridization assays, the sample is typicallycontacted with an array under stringent hybridization conditions,whereby complexes are formed between target nucleic acids that agent arecomplementary to probe sequences attached to the array surface, i.e.,duplex nucleic acids are formed on the surface of the substrate by theinteraction of the probe nucleic acid and its complement target nucleicacid present in the sample. A “stringent hybridization” and “stringenthybridization wash conditions” in the context of nucleic acidhybridization (e.g., as in array, Southern or Northern hybridizations)are sequence dependent, and are different under different experimentalparameters. Stringent hybridization conditions that can be used toidentify nucleic acids within the scope of the invention can include,e.g., hybridization in a buffer comprising 50% formamide, 5×SSC, and 1%SDS at 42° C., or hybridization in a buffer comprising 5×SSC and 1% SDSat 65° C., both with a wash of 0.2×SSC and 0.1% SDS at 65° C. Exemplarystringent hybridization conditions can also include a hybridization in abuffer of 40% formamide, 1 M NaCl, and 1% SDS at 37° C., and a wash in1×SSC at 45° C. Alternatively, hybridization to filter-bound DNA in 0.5M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. can be employed. Yet additionalstringent hybridization conditions include hybridization at 60° C. orhigher and 3×SSC (450 mM sodium chloride/45 mM sodium citrate) orincubation at 42° C. in a solution containing 30% formamide, 1M NaCl,0.5% sodium sarcosine, 50 mM MES, pH 6.5. Those of ordinary skill willreadily recognize that alternative but comparable hybridization and washconditions can be utilized to provide conditions of similar stringency.

In certain embodiments, the stringency of the wash conditions that setforth the conditions which determine whether a nucleic acid isspecifically hybridized to a surface bound nucleic acid. Wash conditionsused to identify nucleic acids may include, e.g.: a salt concentrationof about 0.02 molar at pH 7 and a temperature of at least about 50° C.or about 55° C. to about 60° C.; or, a salt concentration of about 0.15M NaCl at 72° C. for about 15 minutes; or, a salt concentration of about0.2×SSC at a temperature of at least about 50° C. or about 55° C. toabout 60° C. for about 15 to about 20 minutes; or, the hybridizationcomplex is washed twice with a solution with a salt concentration ofabout 2×SSC containing 0.1% SDS at room temperature for 15 minutes andthen washed twice by 0.1×SSC containing 0.1% SDS at 68° C. for 15minutes; or, equivalent conditions. Stringent conditions for washing canalso be, e.g., 0.2×SSC/0.1% SDS at 42° C.

A specific example of stringent assay conditions is rotatinghybridization at 65° C. in a salt based hybridization buffer with atotal monovalent cation concentration of 1.5 M (e.g., as described inU.S. patent application Ser. No. 09/655,482 filed on Sep. 5, 2000, thedisclosure of which is herein incorporated by reference) followed bywashes of 0.5×SSC and 0.1×SSC at room temperature.

Stringent assay conditions are hybridization conditions that are atleast as stringent as the above representative conditions, where a givenset of conditions are considered to be at least as stringent ifsubstantially no additional binding complexes that lack sufficientcomplementarity to provide for the desired specificity are produced inthe given set of conditions as compared to the above specificconditions, where by “substantially no more” is meant less than about5-fold more, typically less than about 3-fold more. Other stringenthybridization conditions are known in the art and may also be employed,as appropriate.

The array is incubated with the sample under appropriate array assayconditions, e.g., hybridization conditions, as mentioned above, whereconditions may vary depending on the particular biopolymeric array andbinding pair.

Once the incubation step is complete, the array is typically washed atleast one time to remove any unbound and non-specifically bound samplefrom the substrate, generally at least two wash cycles are used. Washingagents used in array assays are known in the art and, of course, mayvary depending on the particular binding pair used in the particularassay. For example, in those embodiments employing nucleic acidhybridization, washing agents of interest include, but are not limitedto, salt solutions such as sodium, sodium phosphate (SSP) and sodium,sodium chloride (SSC) and the like as is known in the art, at differentconcentrations and which may include some surfactant as well.

Following the washing procedure, the array may then be interrogated orread to detect any resultant surface bound binding pair or target/probecomplexes, e.g., duplex nucleic acids, to obtain signal data related tothe presence of the surface bound binding complexes, i.e., the label isdetected using colorimetric, fluorimetric, chemiluminescent,bioluminescent means or other appropriate means. The obtained signaldata from the reading may be in any convenient form, i.e., may be in rawform or may be in a processed form.

As such, in using an array made by the method of the present invention,the array will typically be exposed to a sample (for example, afluorescently labeled analyte, e.g., protein containing sample) and thearray then read. Reading of the array to obtain signal data may beaccomplished by illuminating the array and reading the location andintensity of resulting fluorescence (if such methodology was employed)at each feature of the array to obtain a result. For example, an arrayscanner may be used for this purpose that is similar to the AgilentMICROARRAY SCANNER available from Agilent Technologies, Palo Alto,Calif. Other suitable apparatus and methods for reading an array toobtain signal data are described in U.S. patent application Serial Nos:Ser. No. 09/846,125 “Reading Multi-Featured Arrays” by Dorsel et al.;and Ser. No. 09/430,214 “Interrogating Multi-Featured Arrays” by Dorselet al., the disclosures of which are herein incorporated by reference.However, arrays may be read by any other method or apparatus than theforegoing, with other reading methods including other optical techniques(for example, detecting chemiluminescent or electroluminescent labels)or electrical techniques (where each feature is provided with anelectrode to detect hybridization at that feature in a manner disclosedin U.S. Pat. No. 6,221,583, the disclosure of which is hereinincorporated by reference, and elsewhere).

One such system for reading an array produced according to the subjectmethods is shown in FIG. 4. which illustrates an array reader at asingle “user station”, which may (but not necessarily) be remote fromthe fabrication station of FIG. 3 (usually the user station is at thelocation of the customer which ordered the fabricated, received array).The user station includes a processor 162, a memory 184, a scanner 160which can read an array, data writer/reader 186 which may be capable ofwriting/reading to the same type of media as writer/reader 326), and acommunication module 164 which also has access to communication channel180. Processor 162 is programmed to perform all the functions requiredof it. Scanner 160 may include a holder 161 which receives and holds anarray assembly, as well as a source of illumination (such as a laser)and one or more light sensors 165 to read fluorescent light signals fromrespective features on the array as signal data which is obtained byprocessor 162 from the light sensor. Scanner 160 also includes a reader163 to read a bar code 356 appearing on an array assembly 15.Processor-162 may also be capable of identifying signal data from readfeatures of a same probe composition with different feature sizes, basedon the read indication from a read bar code, and merging signal datafrom such.

Communication module 164 may be any type of suitable communicationmodule, such as those described in connection with communication module144. Memory 184 can be any type of memory such as those used for memory141. Scanner 160 may be any suitable apparatus for reading an array,such as one which can read the location and intensity of fluorescence ateach feature of an array following exposure to a fluorescently labeledsample. For example, such a scanner may be similar to the MICROARRAYSCANNER available from Agilent Technologies, Inc. Palo Alto, Calif.Other suitable apparatus and methods are described in U.S. patentapplications: Ser. No. 09/846,125 “Reading Multi-Featured Arrays” byDorsel et al.; and U.S. Pat. No. 6,406,849. The scanning components ofscanner 160, holder 161, and reader 163 may all be contained within thesame housing of a single same apparatus.

Regardless of the particular method and apparatus employed to read anarray, information obtained from the array assay relating to the designof the array and/or information about a particular sample or targetthereof may be incorporated into future array designs. Information suchas the number of feature sizes, the sizes of the features, thecompositions of the features, the distances between features, and thesignals obtained from various array designs may be used to makealgorithms used in array fabrication more robust. The subject algorithmsmay be iterated, each time changing one or more of the array designparameters, e.g., based on previous array assay results, until thesignals obtained from an array meets predetermined criteria such asdesign specifications and the like.

In certain embodiments, the results of the array reading (processed ornot) may be forwarded (such as by communication) to a remote location ifdesired, and received there for further use (such as furtherprocessing). By “remote location” is meant a location other than thelocation at which the sample evaluation device is present and sampleevaluation occurs. For example, a remote location could be anotherlocation (e.g., office, lab, etc.) in the same city, another location ina different city, another location in a different state, anotherlocation in a different country, etc. As such, when one item isindicated as being “remote” from another, what is meant is that the twoitems are at least in different buildings, and may be at least one mile,ten miles, or at least one hundred miles apart. “Communicating”information means transmitting the data representing that information aselectrical signals over a suitable communication channel (for example, aprivate or public network). “Forwarding” an item refers to any means ofgetting that item from one location to the next, whether by physicallytransporting that item or otherwise (where that is possible) andincludes, at least in the case of data, physically transporting a mediumcarrying the data or communicating the data. The data may be transmittedto the remote location for further evaluation and/or use. Any convenienttelecommunications means may be employed for transmitting the data,e.g., facsimile, modem, Internet, etc.

As noted above, the arrays produced according to the subject method maybe employed in a variety of array assays including hybridization assays.Specific hybridization assays of interest which may be practiced usingthe subject arrays include: gene discovery assays, differential geneexpression analysis assays; nucleic acid sequencing assays, and thelike. Patents describing methods of using arrays in various applicationsinclude: U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049;5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839;5,580,732; 5,661,028; 5,800,992; the disclosures of which are hereinincorporated by reference.

Other array assays of interest include those where the arrays are arraysof polypeptide binding agents, e.g., protein arrays, where specificapplications of interest include analyte detection/proteomicsapplications, including those described in U.S. Pat. Nos. 4,591,570;5,171,695; 5,436,170; 5,486,452; 5,532,128; and 6,197,599; as well aspublished PCT application Nos. WO 99/39210; WO 00/04832; WO 00/04389; WO00/04390; WO 00/54046; WO 00/63701; WO 01/14425; and WO 01/40803; thedisclosures of the United States priority documents of which are hereinincorporated by reference.

For example, embodiments may include using an array prepared accordingto the subject methods (e.g., an array having two or more differentsized features) at the user station of FIG. 4, by receiving a package340 from the remote fabrication station and opening it to retrieve theprepared array and portable storage medium 324 b (if present in package340). Sample, for example a test sample, may be exposed to the one ormore received arrays in a known manner under known conditions. Apparatusand procedures for hybridization are described, for example, in U.S.Pat. No. 6,258,593 and U.S. Pat. No. 6,399,394, the disclosures of whichare herein incorporated by reference. Following hybridization andwashing, the array may then be inserted into holder 161 in scanner 160and read by it to obtain read results (such as signal data representingthe fluorescence pattern on the array 12). The reader 163 in scanner 160may also read the identifier 356 in association with the correspondingarray, while the array is still positioned in retained in holder 161 orbeforehand. Using identifier 356, processor 162 may then retrieve thecharacteristic data such as the sizes of the features and, e.g., therelative addresses and compositions thereof, for one or more of thearrays from portable storage medium 324 b or from the database of suchinformation in memory 141.

The resulting retrieved characteristic data for an array, e.g., featuresizes, may be used to either control reading of the array or to processinformation obtained from reading the array. For example, the customermay decide (through providing suitable instructions to processor 162)that a particular feature need not be read or the data from reading thatfeature may be discarded, since the polynucleotide sequence at thatfeature is not likely to produce any reliable data under the conditionsof a particular sample hybridization.

Computer Readable Medium Having an Algorithm Stored Thereon

Also provided by the subject invention are algorithms stored on computerreadable medium. The subject algorithms may be employed in the practiceof the subject invention. For example, embodiments include algorithmsfor preparing an array in accordance with the subject invention, e.g.,receiving information about feature sizes and/or particular samples tobe used with the array and determining appropriate array parameters suchas applied activation signal for each ejector based on this informationand/or directing a fluid drop deposition device to fabricate an arrayaccording to such information, e.g., by applying suitable waveforms toeach ejector based on the desired feature sizes.

More specifically, arrays may be designed manually or with theassistance of a computing means, in which an algorithm is employed thatis capable of directing suitable software/hardware means to prepare anarray with respect to the desired feature sizes of the array, e.g.,based on the amount of target present or at least suspected of beingpresent in a sample. Typically, the algorithm is recorded on a computerreadable storage medium, where such media are well known to those ofskill in the art. More specifically, one or more aspects of the subjectinvention may be in the form of computer readable media having analgorithm, e.g., computer programming, stored thereon for implementingsome or all of the subject methods. For example, the sizes of thefeatures and/or corresponding applied activation signals to providecertain feature sizes of an array may be determined using an algorithmin conjunction with a computational analysis system.

Accordingly, embodiments of the subject invention include computerreadable media having programming (also known as computer control logic)stored thereon for implementing the steps required to determine theappropriate waveform for each ejector of a deposition head, e.g., priorto activation of the ejector and in certain embodiment prior to eachactivation. The computer readable media may be, for example, in the formof a computer disk or CD, a floppy disc, a magnetic “hard card”, aserver, or any other computer readable media capable of containing dataor the like, stored electronically, magnetically, optically or by othermeans. Stored programming may be transferred to a computer such as apersonal computer (PC), (i.e., accessible by a researcher or the like),or to an array fabrication device such as a fluid deposition device byphysical transfer of a CD, floppy disk, or like medium, or may betransferred using a computer network, server, or other interfaceconnection, e.g., the Internet.

In certain embodiments of the subject invention, a system of theinvention may include a computer or the like with a stored algorithmcapable of carrying out array design methods, i.e., a computationalanalysis system. In certain embodiments, the system may be furthercharacterized in that it provides a user interface, where the userinterface presents to a user the option of selecting among one or moredifferent, including multiple different, inputs, e.g., various parametervalues for the algorithm. Computational systems that may be readilymodified to become systems of the subject invention include thosedescribed in U.S. Pat. No. 6,251,588; the disclosure of which is hereinincorporated by reference.

Kits

Finally, kits for use in practicing the subject invention are alsoprovided. The subject kits may include one or more chemical arraysfabricated in accordance with the subject methods. Arrays present in akit may have at least two features that differ at least in size. Thedifferent sized features may or may not differ in composition.

The kits may further include one or more additional components necessaryfor carrying out an analyte detection assay, such as sample preparationreagents, buffers, labels, and the like. As such, the kits may includeone or more containers such as vials or bottles, with each containercontaining a separate component for the assay, and reagents for carryingout an array assay such as a nucleic acid hybridization assay or thelike. The kits may also include a denaturation reagent for denaturingthe analyte, buffers such as hybridization buffers, wash mediums, enzymesubstrates, reagents for generating a labeled target sample such as alabeled target nucleic acid sample, negative and positive controls.

In addition to one or more chemical arrays, the subject kits may alsoinclude written instructions for using the chemical arrays in arrayassays such as hybridization assays or protein binding assays. Theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or sub-packaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.,CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the Internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

The subject kit may also include one or more algorithms, as describedabove, present on computer readable medium, or means for accessing suchalgorithms such as means for obtaining the algorithms from a remotesource, e.g. via the Internet. Databases may also be provided, e.g., ona computer readable medium. These databases may include a population ofdifferent waveforms and corresponding feature sizes and/or dropletsizes.

Reagents for fabricating a chemical array according to the subjectinvention may also be provided in a subject kit e.g., one or more of:biopolymers or precursors thereof, buffers, activator fluid, cappingfluids, and the like. As such, the kits may include one or morecontainers such as vials or bottles, with each container containing aseparate component for an array fabrication protocol.

In many embodiments of the subject kits, the components of the kit arepackaged in a kit containment element to make a single, easily handledunit, where the kit containment element, e.g., box or analogousstructure, may or may not be an airtight container, e.g., to furtherpreserve the one or more chemical arrays and reagents, if present, untiluse.

It is evident from the above results and discussion that theabove-described invention provides methods and devices that preciselycontrol the amount of fluid dispensed from each orifice of a fluiddeposition device. Accordingly, methods and devices that are capable offabricating chemical arrays having different sizes are provided. Assuch, the subject invention represents a significant contribution to theart.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of fabricating a chemical array, said method comprising: (a) determining a chemical array layout in which each feature in said chemical array layout has a size that is chosen based on its composition; and (b) fabricating said chemical array according to said chemical array layout.
 2. The method of claim 1, wherein at least two features are of different sizes.
 3. The method of claim 2, wherein said at least two features are of the same probe composition.
 4. The method of claim 2, wherein said at least two features are of different probe compositions.
 5. The method of claim 1, wherein said fabricating is accomplished with a fluid drop deposition device.
 6. The method of claim 5, wherein said fluid drop deposition device comprises at least one deposition head and said fabricating comprises modulating the applied activation signal for each ejector of said at least one deposition head to produce said features.
 7. The method of claim 6, wherein said deposition head is under the control of a processor and said method comprises transmitting said feature sizes to said processor, whereby said processor performs said modulating based on said feature sizes.
 8. The method of claim 7, wherein each ejector is a piezoelectric ejector.
 9. The method of claim 1, wherein said chemical array is a nucleic acid array.
 10. The method of claim 1, wherein said chemical array is a peptide array.
 11. A method of fabricating a chemical array with multiple features of different sizes, said method comprising: modulating a waveform provided to at least one orifice ejector based on said chemical array feature sizes to dispense volumes of fluid from an orifice associated with said at least one orifice ejector, wherein said volume dispensed is based on said modulated waveform provided to said orifice ejector.
 12. The method of claim 11, wherein said method comprises providing a first modulated waveform based on a feature size of a first feature and second modulated waveform based on a feature of a second feature to said at least one ejector orifice.
 13. The method of claim 12, wherein said first and second modulated waveforms are provided to the same orifice ejector which is associated with a single orifice, whereby said volume of fluid for said first feature and said volume of fluid for said second feature are both dispensed from said single orifice.
 14. The method of claim 12, wherein said first and second modulated waveforms are provided to different orifice ejectors associated with different orifices, whereby said volume of fluid for said first feature and said volume of fluid for said second feature are dispensed from different orifices.
 15. The method of claim 11, wherein the feature size of a first feature is different than the feature size of a second feature and said modulation comprises providing a first waveform to said at least one orifice ejector to dispense a first volume of fluid for said first feature and a second waveform to said at least one orifice ejector to dispense a second volume of fluid for said second feature.
 16. The method of claim 15, wherein said first and second waveforms are provide to the same orifice ejector and said first and second volumes of fluid are dispensed from the same orifice.
 17. The method of claim 15, wherein said first and second waveforms are provide to different orifice ejectors and said first and second volumes of fluid are dispensed from different orifices.
 18. The method of claim 11, wherein said modulating step comprises providing an activation signal to said at least one orifice ejector.
 19. The method of claim 18, wherein said method further comprises selecting said activation signal from a database that comprises a population of activation signals and respective feature sizes.
 20. The method of claim 11, wherein at least one of said dispensed fluids is a phosphoramidite fluid.
 21. The method of claim 11, wherein said at least one of said dispensed fluids is an activator fluid.
 22. The method of claim 11, wherein said method is a method of fabricating a nucleic acid array.
 23. The method of claim 11, wherein said method is a method of fabricating a peptide array.
 24. A method of fabricating a chemical array with multiple features if different sizes, said method comprising: modulating a waveform provided to at least one orifice ejector based on said feature sizes to dispense one or more drops of fluid from an orifice associated with said at least one orifice ejector, wherein a volume of each drop dispensed is based on said modulated waveform provided to said orifice ejector.
 25. A method comprising performing an array assay with a chemical array fabricated according to a method of claim
 1. 26. A method comprising transmitting data from a method of claim 25 from a first location to a second location.
 27. The method according to claim 26, wherein said second location is a remote location.
 28. A method comprising receiving a transmitted result of a reading of an array obtained according to the method claim
 25. 29. An algorithm for practicing the method of claim 1, wherein said algorithm is recorded on a computer readable medium.
 30. An apparatus for fabricating a chemical array, wherein said apparatus is capable of fabricating a chemical array wherein all of the features of said chemical array differ in size.
 31. The apparatus of claim 30, wherein said apparatus comprises at least one deposition head comprising a plurality of orifices and each orifice comprises an ejector activated by an activation signal, and further wherein the activation signal provided to each ejector is capable of being modulated prior to activation.
 32. A system for fabricating a chemical array, said system comprising: (a) a fluid drop deposition device for fabricating a chemical array, wherein said fluid drop deposition device is capable of controlling the size of each feature of said array that is fabricated by said apparatus; and (b) at least one chemical array layout.
 33. A kit comprising: (a) a chemical array fabricated according to the method of claim 1, and (b) instructions for using said fabricated chemical array in an array assay. 